GCN4 is a transcriptional activator of amino acid biosynthetic genes in S. cerevisiae that is repressed at the translational level by four short upstream open-reading-frames (uORFs). In cells starved for an amino acid or purine, ribosomes translate uORF1, bypass the start codons at uORFs 2-4, and reinitiate translation at GCN4 instead, because they fail to rebind a ternary complex of initiation factor-2 (eIF-2), GTP and charged initiator tRNAMet until after scanning past uORF4. The concentration of ternary complexes is reduced in starved cells by phosphorylation of the alpha by GCN2. Several lines of evidence indicate that phosphorylated eIF-2 regulates translation in yeast by inhibiting guanine nucleotide exchange on eIF-2 by eIF-2B. Point mutations in the GCN3, GCD7, and GCD2 subunits of eIF-2B uncouple GCN4 translation from eIF-2alpha phosphorylation. These mutations cluster in a region of homology between the three protein that may define the sites of interaction between eIF-2B and eIF-2(alphaP). Overexpression of only these three eIF-2B subunits reverses the effects of eIF-2alpha hyperphosphorylation, suggesting that they form a subcomplex of eIF-2B components that can sequester eIF-2(alphaP). The effects of eIF-2alpha hyperphosphorylation is suppressed by overexpressing the three subunits of eIF-2, even though this leads to higher absolute levels of eIF-2(alphaP), the inhibitor of eIF-2B. This establishes that eIF-2(alphaP) inhibits eIF-2B by competitive inhibition rather than by non-irreversible binding of eIF-2(alphaP) to eIF-2B. GCN4 translation can be induced by decreasing the dosage of initiator tRNAMet genes, providing in vivo evidence that phosphorylation of eIF-2 reduces the level of ternary complexes. In addition to a requirement for A+U-rich sequences surrounding the uORF1 stop codon, translational reinitiation at GCN4 is dependent on sequences in the leader preceding and GCN20 proteins are required in vivo for phosphorylation of eIF-2alpha by GCN2, and GCN1 and GCN20 were found to be components of the same high molecular weight complex. The requirements for dsRNA binding in vitro and translational control in yeast cells by the human dsRNA-activated eIF-2alpha kinase (DAI) strongly coincide, providing in vivo evidence that the level of DAI activation is largely a function of its ability to bind dsRNA. The identification of dominant-negative alleles and interallelic complementation involving different DAI mutants indicates that the active form of DAI in yeast cells is a homodimer. The transcriptional activation domain of GCN4 contains two large subdomains, each of which is sufficient for nearly wild-type transcriptional activation in vivo and is dependent on the co-activator ADA2. Each subdomain contains several smaller units, consisting of bulky hydrophobic residues surrounded by acidic amino acids, that cooperate in different combinations to activate transcription. Adenine-repressible transcriptional activation in yeast has been conferred on the CYC1 promoter with an ca. 80 bp fragment from the ADE5,7 gene containing two consensus BAS1 binding sites.